| BackgroundsMagnesium is a biodegradable metallic material, but the degradation rate of pure magnesium is too rapid and produce large amounts of hydrogen, which make the clinical application has been limited. With the the alloy technology’s development, and the application of the methods of surface modification, biodegradable magnesium alloy corrosion resistance and biocompatibility has been greatly improved.Although magnesium alloys have good biocompatibility, but its degradation rate is still faster, to further slow down the degradation rate of the alloy, the scholars have carried out a series of alloy surface modification research, and have formed different types of alloy coating, which not only to delay the degradation, but also improve its biocompatibility. Such as calcium phosphate coating and fluoride coating.But as orthopedic implant materials, that the impact for bone tissue regeneration will decide whether to become a good bone repair materials. The key cells in the bone regeneration process are Osteoblasts and bone marrow mesenchymal stem cells.The biological behavior and osteogenic differentiation on the cells is not yet clear, and the biological behavior of skeletal muscle cells for bone movement is not clear either.In this study intends to conduct further studies to address the problem,with blank or titanium as control, study the osteogenesis biological effects of magnesium alloy, magnesium alloy with calcium phosphate coating and fluorine-coated magnesium alloy. And then clear the nature of the surface of the material,clear impact on the biological behavior of osteoblasts,clear biological effects on skeletal muscle cells, clear serum magnesium and localized bone changes after its implanted, clear the biological behavior and osteogenic differentiation of bone marrow mesenchymal stem cells.Objectives1. Study surface morphology and nature of magnesium alloy and its coating.2. Study the biological behavior of osteoblasts with magnesium alloy and its coating.3. Study the serum magnesium level, material degradation and osteogenic of magnesium alloy and its coating in rabbits.4. Study the biological behavior of skeletal muscle cells with magnesium alloy and its coating.5. Study the biological behavior of bone marrow mesenchymal stem cells with magnesium alloy and its coating.6. Study the osteogenic differentiation of bone marrow mesenchymal stem cells with magnesium alloy and its coating.Methods1. Using scanning electron microscopy to study the surface morphology of magnesium alloy(AZ31N), calcium and phosphorus coating of magnesium alloy(CaP-AZ31B) and fluorine coating magnesium alloy(F-AZ31B), study its elements by energy spectrum. By the magnesium alloy and the coating was immersed in the cell culture medium obtained extracts,then determined the PH value of the extracts,study the protein adsorption capacity of magnesium alloy.2. Cultured osteoblast in extracts, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using Calcein-AM, and ETHD-1double staining to observe cell survival,study phosphatase and total protein of cells in1,3,5,7days.3. There are3groups in implant test:, magnesium alloy group(AZ31B), calcium phosphate coating of magnesium alloy group(CaP-AZ31B), polylactic acid group(PLLA),The pins were implanted into rabbit femur,Then the general behavior of rabbits were observed, serum magnesium concentration of24hours, one week, four weeks, six weeks and eight weeks after operation were measured.The experimental animals were sacrificed after8weeks,using CT scanning and three-dimensional reconstruction of the implant material to study morphological changes, observing histological bone formation,using SEM to observe degradation.4. Cultured skeletal muscle cells in extracts, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using apoptosis detection kit to detect apoptosis, using Calcein-AM, and ETHD-1double staining to observe cell survival,study total protein of cells in1,3,5,7days.5. Bone marrow were drawn from volunteers, followed by the isolation and culture of mesenchymal stem cells in the bone marrow, bone marrow mesenchymal stem cells were identified by immunohistochemistry; through the use of magnesium alloy, calcium and phosphorus coating of magnesium alloy and magnesium fluoride coating alloy extractionliquid cultured bone marrow mesenchymal stem cells, then study the2,6and24hours cell adhesion rate, using scanning electron microscopy to study the adhesion morphology, using the inverted microscope to study cell morphology of1,3,5,7days, CCK assay the osteoblasts proliferation activity of1,3,5,7days, using Calcein-AM, and ETHD-1double staining to observe cell survival.6. Using different osteogenic induction medium induced osteogenic differentiation of bone marrow mesenchymal stem cells, collected by cell primer design cell RNA extraction and reverse transcription process,(3-actin gene as an internal,study the ALP, COL I,OC, OPN and RUNX2gene expression after cells were cultured in various extracts for6days and12days.7. The data was staticstically analysis with one-way ANOVA analysis and Univariate, and LSD test was compared in pairwise comparison. Level of significance α=0.05.Results1. The surface morphology and the nature1.1The surface morphology:Magnesium alloy surface morphology was rough, smooth, calcium phosphate coating magnesium alloy surface morphology seen a lot of calcium and phosphorus material deposition and crystal-like structure, fluorine coating the surface of the magnesium alloy surface is uniform, smooth, visible distribution of a small number of irregular holes in the surface of the coating.1.2EDS analysis:The results showed that the surface of the magnesium alloy are mainly magnesium, aluminum, zinc composition, where in the magnesium atomic percentage is approximately96%, and aluminum of about3%, zinc is about1%; calcium phosphate coating magnesium alloy surface of the CaP-AZ31B mainly consists of magnesium, calcium, phosphorus, and oxygen, where in the percentage of magnesium atom of about0.33%, oxygen63.39%,17.6%phosphorus, calcium18.63%; magnesium fluoride coating the surface of the alloy F-AZ31B magnesium,fluorine, aluminum, zinc, magnesium atom percentage of about56%, about42.34%fluorine, aluminum is about1.27%, zinc is about0.47%.1.3PH value changes of extracts:The PH of AZ31B and its coating extracts changed gradually to alkalienvironmental transformation, but the PH value of the magnesium alloy AZ31B extraction of liquid increased most significantly, no significant changes in the solution pH value of the F-AZ31B, CaP-AZ31B solution PH value change between AZ31B and F-AZ31B.1.4Protein adsorption ability:magnesium alloy protein adsorption capacity is lower than the titanium alloy low, the protein adsorption capacity of F-AZ31B is slightly higher than magnesium alloy, calcium phosphate coating magnesium alloy protein adsorption capacity was significantly higher compared to the other three groups (P<0.05),Protein adsorption capacity of four groups were26.95±3.17,25.27±2.02,70.2±5.57,27.70±2.00ug/ml.2、The biological behavior of osteoblasts2.1Early cell adhesion rateThe cell adhesion rate of MC3T3-E1cells in2h,6h and24h:titanium alloy group (27.58±1.11)%,(34.92±2.26)%,(50.21±2.11)%; magnesium alloy group (24.04±2.33)%,(29.08±1.91)%,(37.33±1.49)%; the fluorine coating magnesium alloy (28.46±1.39)%,(35.96±1.18)%,(51.75±1.94)%; calcium phosphate coating magnesium alloys(34.25±1.47)%,(43.71±1.61)%,(62.75±1.97)%; CaP-AZ31B> F-AZ31B group> titanium alloy group> AZ31B group. Significant differences exist between the groups (P<0.05).2.2Cell adhesion morphology Osteoblast adhesion well in three groups of materials, with osteoblasts adherent expand, irregular shape, mostly fusiform, more protruding part of the cell protrusion interconnected.2.3Cell viabilityCell viability cultured in extracts increased with prolonged incubation time and proliferative activity of the the F-AZ31B group> control group> CaP-AZ31B group> AZ31B group, significant differences exist between the two groups (P<0.05)。2.4Relative growth rateThe relative growth rate of AZ31B was less than80%at four time points, with a toxicity rating of2, the relative growth of CaP-AZ31B>80%, toxicity rating of1, the fluorine coating magnesium alloy group>100%, toxicity rating of0. Significant differences exist between the groups (P<0.05).2.5Staininga small amount of stained red cells could be seen in AZ31B extracts,but the vast majority of cells are still active (green dye), no obvious red dye could be seen in cell culture extracts of calcium and phosphorus coating magnesium alloy and magnesium fluoride coating alloy group.2.6Alkaline phosphatase quantitativeWith prolonged incubation time, the group of alkaline phosphatase expression showed an upward trend, the four groups of alkaline phosphatase expression order the CaP-AZ31B group> F-AZ31B group> control group> AZ31B group, significant differences exist between the two groups (P<0.05)。2.7intracellular total proteinWith the prolonged incubation time the total amount of protein in the cells in each group increased, the amount of protein of the four groups of1,3,5,7, d,blank control group,(60.33±2.38)ug/ml,(98.34±2.19)ug/ml,(122.41±3.92)ug/ml, (148.52±3.62)ug/ml; magnesium alloy group (46.5±1.92) ug/ml,(75.73±2.22) ug/ml,(92.29±3.09) ug/ml,(111.31±3.67)ug/ml; the F-AZ31B group (64.66±1.35) ug/ml,(105.71±1.85) ug/ml,(130.97±2.36)ug/ml, and (160.47±2.41) ug/ml; CaP-the AZ31B group(66.24±1.7) ug/ml,(108.61±1.61) ug/ml,(134.01±3.87) ug/ml,(163.03±3.84) ug/ml. Significant differences exist between the groups (P<0.05).3. Bone implantation experiments3.1postoperative rabbits generally in good condition, postoperative serum magnesium for each time point were within the normal range.3.2Polylactic acid almost completely degraded after implanted eight weeks, and fitting close with the new bone. AZ31B and CaP-AZ31B basically maintain the complete form, which shows little degradation of AZ31B bone outside part, the boundary is not clear, irregular, but clear boundary was seen in CaP-AZ31B and did not see the obvious degradation traces.3.3New bone formation could be seen in CaP-AZ31B group and bone trabeculae compact and rules. The formation of new bone is less in AZ31B group. The sem showed that the edge of AZ31B is irregular, which means degradation has occurred, the edge shape of CaP-AZ31B with preimplantation change is very small.4、The biological behavior of skeletal muscle cells4.1Early cell adhesion rate of skeletal muscle cells:With the prolonged adhesion time, adhesion rate of skeletal muscle cell increased in all groups. The cell adhesion rate of skeletal muscle cells in2h,6h and24h:titanium alloy group (26.46±2.33)%,(34.92±2.26)%,(47.21±2.11)%; magnesium alloy group (23.88±2.90)%,(28.75±2.46)%,(36.79±1.66)%; the fluorine coating magnesium alloy (27.80±2.09)%,(35.33±1.49)%,(49.33±1.42)%; calcium phosphate coating magnesium alloys(33.04±2.69)%、(42.58±1.72)%、(59.29±1.62)%.Significant differences exist between the groups (P<0.05). 4.2Cell adhesion morphologyskeletal muscle cells adhesion well in three groups of materials, with skeletal muscle cells adherent expand, mostly fusiform, more protruding part of the cell protrusion interconnected.4.3Cell viabilityCell viability cultured in extracts increased with prolonged incubation time and proliferative activity of the control group> CaP-AZ31B group>F-AZ31B group> AZ31B group, significant differences exist between the two groups (P<0.05)。4.4Relative growth rateThe relative growth rate of AZ31B was less than80%at5and7days, with a toxicity rating of2, the relative growth of CaP-AZ31B and F-AZ31B>80%, toxicity rating of1, toxicity rating of0. Significant differences exist between the groups (P <0.05).4.5Detection of apoptosisMagnesium alloy extracts could cause skeletal muscle apoptosis, no significant apoptosis were found in the control group and CaP-AZ31B group and F-AZ31B group.4.6Staininga small amount of stained red cells could be seen in AZ31B extracts,but the vast majority of cells are still active (green dye), no obvious red dye could be seen in cell culture extracts of calcium and phosphorus coating magnesium alloy and magnesium fluoride coating alloy group.4.7intracellular total proteinWith the prolonged incubation time the total amount of protein in the cells in each group increased, the amount of protein of the four groups of1,3,5,7, d:blank control group,(75.33±2.72) ug/ml,(125.73±1.993) ug/ml,(163.27±4.197) ug/ml,(199.067±3.528) ug/ml, magnesium alloy group (60.77±2.274) ug/ml,(99.21±2.45) ug/ml,(127.35±3.214) ug/ml,(151.84±3.72) ug/ml, the F-AZ31B group (69.98±3.295)ug/ml,(116.55±3.981) ug/ml,(149.57±4.267) ug/ml,(181.89±7.79)ug/ml, CaP-the AZ31B group (70.52±3.862)ug/ml,(117.17±4.543) ug/ml,(151.68±5.429) ug/ml,(187.31±7.16) ug/ml.Significant differences exist between the groups (P<0.05).5. The biological behavior of bone marrow mesenchymal stem cells5.1Identification of mesenchymal stem cells in the bone marrowFlow cytometry cell cytometry results suggest that CD34-, CD45-, CD44+, CD73+,CD90+,CD105+.5.2Early cell adhesion rate of bone marrow mesenchymal stem cellsWith the prolonged adhesion time, adhesion rate of skeletal muscle cell increased in all groups, but cell adhesion was poor in magnesium alloy surface.The cell adhesion rate of bone marrow mesenchymal stem cells in2h,6h and24h:titanium alloy group(27.08±1.95)%,(35.38±1.93)%,(49.04±2.10)%,magnesium alloy group (20.58±2.20)%,(24.63±2.03)%,(30.83±1.45)%,the fluorine coating magnesium alloy(27.58±2.29)%,(35.88±2.14)%,(50.33±2.70)%, calcium phosphate coating magnesium alloys(30.38±2.31)%、(42.58±1.72)%、(59.29±1.62)%.Significant differences exist between the groups (P<0.05).5.3Cell viabilityCell viability cultured in extracts increased with prolonged incubation time, and proliferative capacity of magnesium alloys was relatively weak, CaP-AZ31B group and F-AZ31B group was relatively good,and the cell proliferation of adjust the PH value group magnesium alloy extracts was good too. Cell staining showed that no obvious red cells could be seen in all group.6、Osteogenic differentiation of bone marrow mesenchymal stem cells RNA extracted more complete, and high purity. RT-PCR results suggest that: CaP-AZ31B could promote the expression of COLI gene, ALP gene and OC gene.No significant difference in the RUNX2gene expression was found among the four groups.ALP expression levels of magnesium alloy group was low, after adjust the PH value,the expression of ALP Promoted and no significant difference was found between blank control group (p>0.05).OPN gene expression levels of magnesium alloy was significantly higher than that of the other three groups after the induction of differentiation6days and12days(p<0.05), no significant difference was found between blank control group after adjust the PH value (P>0.05).which means that PH value could cause the ALP genes and OPN gene expression change.Conclusions11.The surface of AZ31B and F-AZ31B is smooth, but the surface of CaP-AZ31B is roughness with crystal-like structure.2.PH value increased in the three groups extracts, but the fluorine coating magnesium alloy is stable, the PH value of magnesium alloy increased significantly.3.Calcium phosphate coating magnesium alloys have better adsorption capacity for protein, there is no significant difference in protein adsorption among magnesium alloy and titanium alloy and magnesium alloy with fluorine coating.4.Magnesium alloy have toxicity on osteoblast, but F-AZ31B and CaP-AZ31B have good biocompatibility for osteoblast.5.Magnesium alloy and its calcium phosphate coating have good biocompatibility in vivo. Postoperative serum magnesium for each time point were within the normal range. Calcium phosphate coating effectively retard the degradation of magnesium alloy, and can promote bone formation. 6.Magnesium alloy have toxicity on skeletal muscle cells, but F-AZ31B and CaP-AZ31B have good biocompatibility for skeletal muscle cells.7.Magnesium alloy have toxicity on bone marrow mesenchymal stem cells, but F-AZ31B and CaP-AZ31B have good biocompatibility for bone marrow mesenchymal stem cells.8.Calcium phosphate coating of magnesium alloy can promote the expression of COLI, ALP and OC gene,the role of magnesium alloy for the ALP and OPN gene expression is related to the PH value. |
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